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The pollen tube is ideal for studying cell polar growth, and observing the ultrastructure of the pollen tube tip using transmission electron microscopy (TEM) is the primary method for studying pollen tube growth. The preparation of ultrathin sections of the pollen tube tip sample is important for its successful microscopic observation. The direction of pollen tube growth in vitro is irregular, and it is difficult to dissect the tip of the pollen tube during ultrathin sectioning. Here, we used two methods to efficiently obtain an ultrathin section of the pollen tube tip of Pyrus. In the first method, laser micro-cutting was used to obtain the pollen tube tip, followed by ultrathin sectioning. In the other method, the pollen tubes were cultured in the same growth direction, followed by ultrathin sectioning. Ultrathin sections, which were observed via TEM, showed typical characteristics of the pollen tube tip, such as dense vesicles, numerous mitochondria, and secretory vesicles of the Golgi. We concluded that these two methods are effective in pollen tube tip sample preparation for ultrathin sectioning and provide the foundation for observing the ultrastructure of pollen tube tips.

The movement of organelles and vesicles in pollen tubes depends on F-actin. However, the molecular mechanism through which plant myosin XI drives the movement of organelles is still controversial, and the relationship between myosin XI and vesicle movement in pollen tubes is also unclear. In this study, we found that the siliques of the myosin xi-b/e mutant were obviously shorter than those of the wild-type (WT) and that the seed set of the mutant was severely deficient. The pollen tube growth of myosin xi-b/e was significantly inhibited both in vitro and in vivo. Fluorescence recovery after photobleaching (FRAP) showed that the velocity of vesicle movement in the pollen tube tip of the myosin xi-b/e mutant was lower than that of the WT. It was also found that peroxisome movement was significantly inhibited in the pollen tubes of the myosin xi-b/e mutant, while the velocities of the Golgi stack and mitochondrial movement decreased relatively less in the pollen tubes of the mutant. The endoplasmic reticulum streaming in the pollen tube shanks was not significantly different between the WT and the myosin xi-b/e mutant. In addition, we found that myosin XI-B-GFP colocalized obviously with vesicles and peroxisomes in the pollen tubes of Arabidopsis. Taken together, these results indicate that myosin XI-B may bind mainly to vesicles and peroxisomes and drive their movement in pollen tubes. These results also suggest that the mechanism by which myosin XI drives organelle movement in plant cells may be evolutionarily conserved compared with other eukaryotic cells.

While rhodopsin-based optogenetics has revolutionized neuroscience1,2, poor expression of opsins and the absence of the essential cofactor all-trans-retinal has complicated the application of rhodopsins in plants. Here, we demonstrate retinal production in plants and improved rhodopsin targeting for green light manipulation of plant cells using the Guillardia theta light-gated anion channelrhodopsin GtACR13. Green light induces a massive increase in anion permeability and pronounced membrane potential changes when GtACR1 is expressed, enabling non-invasive manipulation of plant growth and leaf development. Using light-driven anion loss, we could mimic drought conditions and bring about leaf wilting despite sufficient water supply. Expressed in pollen tubes, global GtACR1 activation triggers membrane potential depolarizations due to large anion currents. While global illumination was associated with a reversible growth arrest, local GtACR1 activation at the flanks of the apical dome steers growth direction away from the side with increased anion conductance. These results suggest a crucial role of anion permeability for the guidance of pollen tube tip growth. This plant optogenetic approach could be expanded to create an entire pallet of rhodopsin-based tools4, greatly facilitating dissection of plant ion-signalling pathways. The expression of a light-gated anion channelrhodopsin from a unicellular algae and a retinal-producing enzyme from a marine bacterium enable green light optogenetic manipulation of plant cells, as demonstrated in pollen tubes.

Recent studies have shown that loss of pollen-S function in S4′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S4-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S4-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S4-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S4-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of wit12 and wifi, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ peaks in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.

The aim of the current study was to examine the effect of different exogenous putrescine concentrations (200, 400, 600, and 800 μM) on the tea pollen performance. It was shown that putrescine has a dose-dependent effect on pollen performance. Results exhibited that pollen germination and tube elongation were induced by 200 and 400 μM putrescine treatment, especially, 400 μM putrescine-enhanced pollen performance. However, pollen performance was inhibited by higher concentrations of putrescine. Putrescine concentrations above 400 μM changed the actin filament distribution in pollen tubes by affecting the distribution of sucrose synthase enzyme. Alterations of the distribution on sucrose synthase enzyme also caused the alterations in the dispersion of cellulose and callose in the cell wall, and morphological alterations such as balloon-shaped and snake-shaped pollen tube tip accompanied them. Moreover, putrescine concentrations above 400 μM caused a decrease of ROS level in apex and led to chromatin condensation of the generative nucleus. In conclusion, exogenous putrescine application can be used as a pollen performance enhancer at low concentrations while the high concentrations cause adverse effects reducing fertilization success.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

Pectin methylesterase inhibitor gene family in the seven Rosaceae species (including three pear cultivars) is characterized and three pectin methylesterase inhibitor genes are identified to regulate pollen tube growth in pear. Pectin methylesterase inhibitor (PMEI) participates in a variety of biological processes in plants. However, the information and function of PMEI genes in Rosaceae are largely unknown. In this study, a total of 423 PMEI genes are identified in the genomes of seven Rosaceae species. The PMEI genes in pear are categorized into five subfamilies based on structural analysis and evolutionary analysis. WGD and TD are the main duplication events in the PMEI gene family of pear. Quantitative real-time PCR analysis indicates that PbrPMEI23, PbrPMEI39, and PbrPMEI41 are increasingly expressed during pear pollen tube growth. Under the treatment of recombinant proteins PbrPMEI23, PbrPMEI39 or PbrPMEI41, the content of methylesterified pectin at the region 5-20 μm from the pollen tube tip significantly increases, and the growth of pear pollen tubes is promoted. These results indicate that PMEI regulates the growth of pollen tubes by changing the distribution of methylesterified pectin in the apex.

The role of ArabinoGalactan Proteins (AGPs) in the sexual reproduction of gymnosperms is not as well documented as that of angiosperms. In earlier studies, we demonstrated that AGPs play important roles during ovule differentiation in Larix decidua Mill. The presented results encouraged us to carry out further studies focused on the functions of these unique glycoproteins during pollen/pollen tube and ovule interactions in Larix. We identified and analyzed the localization of AGPs epitopes by JIM4, JIM8, JIM13 and LM2 antibodies (Abs) in male gametophytes and ovule tissue during pollination, the progamic phase, and after fertilization and in vitro growing pollen tubes. Our results indicated that (1) AGPs recognized by JIM4 Abs play an essential role in the interaction of male gametophytes and ovules because their appearance in ovule cells is induced by physical contact between reproductive partners, (2) after pollination, AGPs are secreted from the pollen cytoplasm into the pollen wall and contact the extracellular matrix of stigmatic tip cells followed by micropylar canal cells, (3) AGPs synthesized in nucellus cells before pollen grain germination are secreted during pollen tube growth into the extracellular matrix, where they can directly interact with male gametophytes, (4) in vitro cultured pollen tube AGPs labeled with LM2 Abs participate in the germination of pollen grain, while AGPs recognized by JIM8 Abs are essential for pollen tube tip growth.

In flowering plants, sexual reproduction involves a double fertilization event, which is facilitated by the delivery of two non-motile sperm cells to the ovule by the pollen tube. Pollen tube growth occurs exclusively at the tip and is extremely rapid. It strictly depends on an intact actin cytoskeleton, and is therefore an excellent model for uncovering the molecular mechanisms underlying dynamic actin cytoskeleton remodeling. There has been a long-term debate about the organization and dynamics of actin filaments within the apical and subapical regions of pollen tube tips. By combining state-of-the-art live-cell imaging with the usage of mutants which lack different actin-binding proteins, our understanding of the origin, spatial organization, dynamics and regulation of actin filaments within the pollen tube tip has greatly improved. In this review article, we will summarize the progress made in this area.

The growing pollen tube has become one of the most fascinating model cell systems for investigations into cell polarity and polar cell growth in plants. Rapidly growing pollen tubes achieve tip-focused cell expansion by vigorous anterograde exocytosis, through which various newly synthesized macromolecules are directionally transported and deposited at the cell apex. Meanwhile, active retrograde endocytosis counter balances the exocytosis at the tip which is believed to recycle the excessive exocytic components for multiple rounds of secretion. Therefore, apical exocytosis and endocytosis are the frontline cellular processes which drive the polar growth of pollen tubes, although they represent opposite vesicular trafficking events with distinct underpinning mechanisms. Nevertheless, the molecular basis governing the spatiotemporal crosstalk and counterbalance of exocytosis and endocytosis during pollen tube polarization and growth remains elusive. Here we discuss recent insight into exocytosis and endocytosis in sculpturing high rates of polarized pollen tube growth. In addition, we especially introduce the novel integration of mathematical modeling in uncovering the mysteries of cell polarity and polar cell growth.

Efficient transport and delivery of sperm cells (SCs) is vital for angiosperm plant fertility. In Arabidopsis thaliana, SCs are transported through the growing pollen tube by a connection with the vegetative nucleus (VN). During pollen tube growth, the VN leads the way and maintains a fixed distance from the pollen tube tip, while the SCs lag behind the VN. Upon reception at the ovule, the pollen tube bursts and the SCs are released for fertilization. In pollen tubes of Arabidopsis mutants wit12 and wifi, deficient in the outer nuclear membrane component of a plant LINC complex, the SCs precede the VN and the VN falls behind. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ spikes in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ spikes are reduced in response to exogenous ROS, but these spikes are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.SummaryMutants deficient in outer nuclear membrane proteins display defects in reactive oxygen species-induced pollen tube burst and nuclear Ca2+ signatures that correlate with the position of the vegetative nucleus.

Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated. Apple pollen grains were cultured under different concentrations of B. Scanning ion-selective electrode technique, fluorescence labelling and Fourier-transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca2+ concentration ([Ca2+ ]cyt), actin filaments and cell wall components of pollen tubes. B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca2+ influx and thus led to increased [Ca2+ ]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring-like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls. B appears to have an active role in pollen tube growth by affecting [Ca2+ ]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

Many receptor-like kinases localized at the pollen tube tip precisely control tube functions in flowering plants. Two recent reports have identified autocrine peptide ligands and receptor systems, providing insight into the molecular machinery that controls pollen tube growth and termination.

Pollen tubes are tip-growing plant cells that deliver the sperm cells to the ovules for double fertilization of the egg cell and the endosperm. Various directional cues can trigger the reorientation of pollen tube growth direction on their passage through the female tissues. Among the external stimuli, protons serve an important, regulatory role in the control of pollen tube growth. The generation of local guidance cues has been challenging when investigating the mechanisms of perception and processing of such directional triggers in pollen tubes. Here, we developed and characterized a microelectrode device to generate a local proton gradient and proton flux through water electrolysis. We confirmed that the cytoplasmic pH of pollen tubes varied with environmental pH change. Depending on the position of the pollen tube tip relative to the proton gradient, we observed alterations in the growth behavior, such as bursting at the tip, change in growth direction, or complete growth arrest. Bursting and growth arrest support the hypothesis that changes in the extracellular H+ concentration may interfere with cell wall integrity and actin polymerization at the growing tip. A change in growth direction for some pollen tubes implies that they can perceive the local proton gradient and respond to it. We also showed that the growth rate is directly correlated with the extracellular pH in the tip region. Our microelectrode approach provides a simple method to generate protons and investigate their effect on plant cell growth.

F-actin and myosin XI play important roles in plant organelle movement. A few myosin XI genes in the genome of Arabidopsis are mainly expressed in mature pollen, which suggests that they may play a crucial role in pollen germination and pollen tube tip growth. In this study, a genetic complementation assay was conducted in a myosin xi-c (myo11c1) myosin xi-e (myo11c2) double mutant, and fluorescence labeling combined with microscopic observation was applied. We found that myosin XI-E (Myo11C2)-green fluorescent protein (GFP) restored the slow pollen tube growth and seed deficiency phenotypes of the myo11c1 myo11c2 double mutant and Myo11C2-GFP partially colocalized with mitochondria, peroxisomes and Golgi stacks. Furthermore, decreased mitochondrial movement and subapical accumulation were detected in myo11c1 myo11c2 double mutant pollen tubes. Fluorescence recovery after photobleaching experiments showed that the fluorescence recoveries of GFP-RabA4d and AtPRK1-GFP at the pollen tube tip of the myo11c1 myo11c2 double mutant were lower than those of the wild type were after photobleaching. These results suggest that Myo11C2 may be associated with mitochondria, peroxisomes and Golgi stacks, and play a crucial role in organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana.

Pollen development, pollen grain germination and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to fertilize ovules. Pollen grains undergo two major developmental switches: dehydration characterized by metabolic quiescent state, and rehydration upon pollination that leads to extraordinary metabolic and membrane trafficking activity, resulting in germination and rapid tip growth of pollen tubes. To sustain these processes, many plant housekeeping genes evolved their pollen-specific paralogs. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we describe that EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in Arabidopsis pollen, which governs the conventional secretory function of the exocyst, analogically to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination and pollen tube growth. GFP:EXO70A2 was localized similarly to other exocyst subunits to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for the EXO70A1 function in the sporophyte, indicating functional redundancy of these two closely related isoforms. Phylogenetic analysis revealed that the ancient duplication of EXO70A to two (or more) paralogs, one of which is highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in the Arabidopsis pollen required for efficient plant sexual reproduction.

Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction.

International audience; Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.

Tip-focused accumulation of reactive oxygen species (ROS) is tightly associated with pollen tube growth and is thus critical for fertilization. However, it is unclear how tip-growing cells establish such specific ROS localization. Polyamines have been proposed to function in tip growth as precursors of the ROS, hydrogen peroxide. The ABC transporter AtABCG28 may regulate ROS status, as it contains multiple cysteine residues, a characteristic of proteins involved in ROS homeostasis. In this study, we found that AtABCG28 was specifically expressed in the mature pollen grains and pollen tubes. AtABCG28 was localized to secretory vesicles inside the pollen tube that moved toward and fused with the plasma membrane of the pollen tube tip. Knocking out AtABCG28 resulted in defective pollen tube growth, failure to localize polyamine and ROS to the growing pollen tube tip, and complete male sterility, whereas ectopic expression of this gene in root hair could recover ROS accumulation at the tip and improved the growth under high-pH conditions, which normally prevent ROS accumulation and tip growth. Together, these data suggest that AtABCG28 is critical for localizing polyamine and ROS at the growing tip. In addition, this function of AtABCG28 is likely to protect the pollen tube from the cytotoxicity of polyamine and contribute to the delivery of polyamine to the growing tip for incorporation into the expanding cell wall.

International audience; Background: During sexual reproduction, pollen grains land on the stigma, rehydrate and produce pollen tubes that grow through the female transmitting-tract tissue allowing the delivery of the two sperm cells to the ovule and the production of healthy seeds. Because pollen tubes are single cells that expand by tip-polarized growth, they represent a good model to study the growth dynamics, cell wall deposition and intracellular machineries. Aiming to understand this complex machinery, we used a low throughput chemical screen approach in order to isolate new tip-growth disruptors. The effect of a chemical inhibitor of monogalactosyldiacylglycerol synthases, galvestine-1, was also investigated. The present work further characterizes their effects on the tip-growth and intracellular dynamics of pollen tubes. Results: Two small compounds among 258 were isolated based on their abilities to perturb pollen tube growth. They were found to disrupt in vitro pollen tube growth of tobacco, tomato and Arabidopsis thaliana. We show that these 3 compounds induced abnormal phenotypes (bulging and/or enlarged pollen tubes) and reduced pollen tube length in a dose dependent manner. Pollen germination was significantly reduced after treatment with the two compounds isolated from the screen. They also affected cell wall material deposition in pollen tubes. The compounds decreased anion superoxide accumulation, disorganized actin filaments and RIC4 dynamics suggesting that they may affect vesicular trafficking at the pollen tube tip.Results: Two small compounds among 258 were isolated based on their abilities to perturb pollen tube growth. They were found to disrupt in vitro pollen tube growth of tobacco, tomato and Arabidopsis thaliana. We show that these 3 compounds induced abnormal phenotypes (bulging and/or enlarged pollen tubes) and reduced pollen tube length in a dose dependent manner. Pollen germination was significantly reduced after treatment with the two compounds isolated from the screen. They also affected cell wall material deposition in pollen tubes. The compounds decreased anion superoxide accumulation, disorganized actin filaments and RIC4 dynamics suggesting that they may affect vesicular trafficking at the pollen tube tip.Conclusion: These molecules may alter directly or indirectly ROP1 activity, a key regulator of pollen tube growth and vesicular trafficking and therefore represent good tools to further study cellular dynamics during polarized-cell growth.

Manganese (Mn) serves as an essential cofactor for many enzymes in various compartments of a plant cell. Allocation of Mn among various organelles thus plays a central role in Mn homeostasis to support metabolic processes. We report the identification of a Golgi-localized Mn transporter (named PML3) that is essential for rapid cell elongation in young tissues such as emerging leaves and the pollen tube. In particular, the pollen tube defect in the pml3 loss-of-function mutant caused severe reduction in seed yield, a critical agronomic trait. Further analysis suggested that a loss of pectin deposition in the pollen tube might cause the pollen tube to burst and slow its elongation, leading to decreased male fertility. As the Golgi apparatus serves as the major hub for biosynthesis and modification of cell-wall components, PML3 may function in Mn homeostasis of this organelle, thereby controlling metabolic and/or trafficking processes required for pectin deposition in rapidly elongating cells.

Pollen tube guidance in flowering plants is a unique and critical process for successful sexual reproduction. The pollen tube that grows from pollen, which is the male gametophyte, precisely navigates to the embryo sac, which is the female gametophyte, within the pistil. Recent advances have clarified the molecular framework of gametophytic pollen tube guidance. Multiple species-specific attractant peptides are secreted from synergid cells, the proper development and function of which are regulated by female gametes. Multiple receptor-like kinases on the pollen tube tip are involved in sensing species-specific attractant peptides. In this Update article, recent progress in our understanding of the mechanism of gametophytic pollen tube guidance is reviewed, including attraction by synergid cells, control of pollen tube guidance by female gametes, and directional growth of the pollen tube by directional cue sensing. Future directions in the study of pollen tube guidance also are discussed.

Potassium homeostasis is essential for pollen development and pollen-pistil interactions during the sexual reproduction of flowering plants. Here, we described the role of a Shaker K+ channel, OsAKT1.2, in rice pollen germination and growth. OsAKT1.2 is specifically expressed in the tricellular pollen, mature pollen grains and growing pollen tubes. Using CRISPR gene editing, we found that knockout lines did not differ from wildtype in vegetative growth, but showed decreased pollen germination rate both in the germination medium and in vivo. OsAKT1.2-GFP fusion protein was localized in the plasma membrane and enriched at the pollen tube tip. OsAKT1.2 could complement the yeast strain which is deficient in K+ intake. These findings suggest that OsAKT1.2 is associated with pollen germination and tube elongation in rice.

The angiosperm pollen tube delivers two sperm cells into the embryo sac through a unique growth strategy, named tip growth, to accomplish fertilization. A great deal of experiments have demonstrated that actin bundles play a pivotal role in pollen tube tip growth. There are two distinct actin bundle populations in pollen tubes: the long, rather thick actin bundles in the shank and the short, highly dynamic bundles near the apex. With the development of imaging techniques over the last decade, great breakthroughs have been made in understanding the function of actin bundles in pollen tubes, especially short subapical actin bundles. Here, we tried to draw an overall picture of the architecture, functions and underlying regulation mechanism of actin bundles in plant pollen tubes.

Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regulation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.

Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca2+ throughout the bulging tip, whereas LaCl3, a Ca2+ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca2+ homeostasis through its potential function in the modulation of Ca2+ influx.

In flowering plants, the interaction of pollen tubes with female tissues is important for the accomplishment of double fertilization. Little information is known about the mechanisms that underlie signalling between pollen tubes and female tissues. In this study, two Arabidopsis pollen tube-expressed CrRLK1L protein kinases, Buddha's Paper Seal 1 (BUPS1) and BUPS2, were identified as being required for normal tip growth of pollen tubes in the pistil. They are expressed prolifically in pollen and pollen tubes and are localized on the plasma membrane of the pollen tube tip region. Mutations in BUPS1 drastically reduced seed set. Most of the bups1 mutant pollen tubes growing in the pistil exhibited a swollen pollen tube tip, leading to failure of fertilization. The bups2 pollen tubes had a slightly abnormal morphology but could still accomplish double fertilization. The bups1 bups2 double mutant exhibited a slightly enhanced phenotype compared to the single bups1 mutants. The BUPS1 proteins could form homomers and heteromers with BUPS2, whereas BUPS2 could only form heteromers with BUPS1. The BUPS proteins could interact with the Arabidopsis pollen-expressed RopGEFs in the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. The results indicated that the BUPSs may mediate normal polar growth of pollen tubes in the pistil.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Pollen tubes elongate rapidly at their tips through highly polarized cell growth known as tip growth. Tip growth requires intensive exocytosis at the tip, which is supported by a dynamic cytoskeleton and vesicle trafficking. Several signaling pathways have been demonstrated to coordinate pollen tube growth by regulating cellular activities such as actin dynamics, exocytosis, and endocytosis. These signaling pathways crosstalk to form a signaling network that coordinates the cellular processes required for tip growth. The homeostasis of key signaling molecules is critical for the proper elongation of the pollen tube tip, and is commonly fine-tuned by positive and negative regulations. In addition to the major signaling pathways, emerging evidence implies the roles of other signals in the regulation of pollen tube growth. Here we review and discuss how these signaling networks modulate the rapid growth of pollen tubes.

Pollen tube guidance is controlled by multiple complex interactions with the female tissues. Here, we show that pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent manner to receive and respond to attractant LURE peptides secreted from synergid cells. We developed an immunostaining method to visualize LURE peptides bound at the plasma membrane of the tip region of the pollen tube. Using this method, we found that LURE peptides bound specifically to pollen tubes growing through a cut style. The peptides also bound to pollen tubes growing through a shorter style, which were not competent to respond to these peptides. These observations suggested a possibility that acquisition of the LURE peptide reception ability and acquisition of full competency are separable processes. RNA-Seq suggested that the transcription profile of pollen tubes was affected by both the length of the style and the cultivation period, consistently with physiological changes in binding activity and LURE response ability. The database generated from de novo RNA-Seq of Torenia pollen tubes was shown to be useful to identify pollen tube proteins by mass spectrometry. Our studies provide insight and an effective platform for protein identification to understand pollen tube guidance.

Summary Successful sexual reproduction in animals and plants requires communication between male and female gametes. In flowering plants, unlike in animals, eggs and sperm cells are enclosed in multicellular embryo sacs and pollen grains, respectively [1]; guided growth of the pollen tube into the ovule is necessary for fertilization [2]. Pollen tube guidance requires accurate perception of ovule-emitted guidance cues by the receptors in pollen tubes [2–4]. Although several ovule-secreted peptides controlling pollen tube guidance have recently been identified, i.e., maize EGG APPARATUS1 (EA1) [5], Torenia LURE1/LURE2 [6], and Arabidopsis CRP810_1/AtLURE1 [7], little is known about the receptors. Here, we identified two receptor-like kinase (RLK) genes preferentially expressed in Arabidopsis pollen tubes, Lost In Pollen tube guidance 1 ( LIP1 ) and 2 ( LIP2 ), which are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control. Simultaneous inactivation of LIP1 and LIP2 led to impaired pollen tube guidance into micropyle and significantly reduced attraction of pollen tubes toward AtLURE1 [7]. Our results suggest that LIP1 and LIP2 represent essential components of the pollen tube receptor complex to perceive the female signal AtLURE1 for micropylar pollen tube guidance.

In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca2+]cyt gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca2+ channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth., Author Summary In the pistils of flowering plants, pollen tubes elongate at the tips to deliver the male gametes to the egg cells for fertilization. The tip growth of pollen tube is due to the deposition of cell membranes and wall materials at a restricted tip area of the plasma membrane. Vesicle trafficking events occurred at the extreme tips have been shown to be required for the coordinated tip growth, while other trafficking pathways are less well characterized. On the other hand, little is known about the molecular mechanisms and the physiological impact of Golgi trafficking and Golgi structure maintenance in plant cells. COG complex is a vesicle tethering factor supposed to mediate intra-Golgi retrograde transport in mammalian and yeast cells. By characterization of two plant COG complex subunits COG3 and COG8, we revealed their roles in Golgi transport and Golgi structure maintenance, which are essential for pollen tube polar growth. These results deepened our understandings on pollen tube growth regulation, and the molecular mechanisms of Golgi trafficking and Golgi morphology maintenance in plant cells.

Nitric oxide (NO) as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca(2+) gradient, increase in ROS content, acidification of cytoplasmic pH and abnormalities in organelle ultrastructure and cell wall component distribution in the pollen tube tip. Furthermore, differentially expressed genes (DEGs)-related to signaling pathway, such as NO synthesis, cGMP, Ca(2+), ROS, pH, actin, cell wall, and MAPK cascade signal pathways, are identified and quantified using transcriptomic analyses and qRT-PCR, which indicate a potential molecular mechanism for the above cytological results. Taken together, these findings suggest that a complex signaling network dominated by NO, including Ca(2+), ROS, pH, RACs signaling and the crosstalk among them, is stimulated in the C. sinensis pollen tube in response to cold stress, which further causes secondary and tertiary alterations, such as ultrastructural abnormalities in organelles and cell wall construction, ultimately resulting in perturbed pollen tube extension.

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3′,5′-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca2+-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response.

Microspores develop inside the anther, where they are surrounded by nourishing tapetal cells. However, many cellular processes occurring during microspore development in the locule are poorly characterized. The actin cytoskeleton is known to play a crucial role in various aspects of the plant developmental process. During pollen tube tip growth, actin cytoskeleton serves as an efficient molecular transportation track, although how it functions in pollen development is unknown. The plant actin bundler PLIM2s have been shown to regulate actin bundling in different cells. Here, we investigate the biological function of three Arabidopsis pollen-specific LIM proteins, PLIM2a, PLIM2b, and PLIM2c (collectively, PLIM2s), in pollen development and tube growth. Variable degrees of suppressed expression of the PLIM2s by RNA interference resulted in aberrant phenotypes. Complete suppression of the PLIM2s totally disrupted pollen development, producing abortive pollen grains and rendering the transgenic plants sterile. Partial suppression of the PLIM2s arrested pollen tube growth to a lesser extent, resulting in short and swollen pollen tubes. Finally, the PLIM2c promoter initiated expression in pollen during stamen filament elongation, and the PLIM2c protein was located on particle structures in the developing pollen grains in Arabidopsis. These suggest that the actin bundler, PLIM2s, are an important factor for Arabidopsis pollen development and tube growth.

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/ Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Salem, T. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Mazzella, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wengier, D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Muschietti, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

During reproduction in flowering plants, pollen grains form a tube that grows in a polarized fashion through the female tissues to eventually fertilize the egg cell. These highly polarized pollen tubes have a rapid rate of growth that is supported by a tip-focused delivery of membrane and cell wall components. To gain a better understanding of how this growth is regulated, we investigated the function RABA4D, a member of the Arabidopsis thaliana RabA4 subfamily of Rab GTPase proteins. Here, we show that RABA4D was expressed in a pollen-specific manner and that enhanced yellow fluorescent protein (EYFP)-RabA4d-labeled membrane compartments localized to the tips of growing pollen tubes. Mutant pollen in which the RABA4D gene was disrupted displayed bulged pollen tubes with a reduced rate of growth in vitro and displayed altered deposition of some cell wall components. Expression of EYFP-RabA4d restored wild-type phenotypes to the raba4d mutant pollen tubes, while expression of EYFP-RabA4b did not rescue the raba4d phenotype. In vivo, disruption of RABA4D resulted in a male-specific transmission defect with mutant raba4d pollen tubes displaying aberrant growth in the ovary and reduced guidance at the micropyle. We propose that RabA4d plays an important role in the regulation of pollen tube tip growth.

Summary In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca2+ ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.

Directional control of tip-growing cells is essential for proper tissue organization and cell-to-cell communication in animals and plants. In the sexual reproduction of flowering plants, the tip growth of the male gametophyte, the pollen tube, is precisely guided by female cues to achieve fertilization. Several female-secreted peptides have recently been identified as species-specific attractants that directly control the direction of pollen tube growth. However, the method by which pollen tubes precisely and promptly respond to the guidance signal from their own species is unknown. Here we show that tip-localized pollen-specific receptor-like kinase 6 (PRK6) with an extracellular leucine-rich repeat domain is an essential receptor for sensing of the LURE1 attractant peptide in Arabidopsis thaliana under semi-in-vivo conditions, and is important for ovule targeting in the pistil. PRK6 interacted with pollen-expressed ROPGEFs (Rho of plant guanine nucleotide-exchange factors), which are important for pollen tube growth through activation of the signalling switch Rho GTPase ROP1 (refs 7, 8). PRK6 conferred responsiveness to AtLURE1 in pollen tubes of the related species Capsella rubella. Furthermore, our genetic and physiological data suggest that PRK6 signalling through ROPGEFs and sensing of AtLURE1 are achieved in cooperation with the other PRK family receptors, PRK1, PRK3 and PRK8. Notably, the tip-focused PRK6 accumulated asymmetrically towards an external AtLURE1 source before reorientation of pollen tube tip growth. These results demonstrate that PRK6 acts as a key membrane receptor for external AtLURE1 attractants, and recruits the core tip-growth machinery, including ROP signalling proteins. This work provides insights into the orchestration of efficient pollen tube growth and species-specific pollen tube attraction by multiple receptors during male-female communication.

ORCID IDs: 0000-0003-1729-0561 (P.B.-V.); 0000-0003-3459-1331 (G.-Y.J.) Pollen tubes are rapidly growing specialized structures that elongate in a polar manner. They play a crucial role in the delivery of sperm cells through the stylar tissues of the flower and into the embryo sac, where the sperm cells are released to fuse with the egg cell and the central cell to give rise to the embryo and the endosperm. Polar growth at the pollen tube tip is believed to result from secretion of materials by membrane trafficking mechanisms. In this study, we report the functional characterization of Arabidopsis BET11 and BET12, two genes that may code for Qc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Double mutants (bet11/bet12) in a homozygous/heterozygous background showed reduced transmission of the mutant alleles, reduced fertilization of seeds, defective embryo development, reduced pollen tube lengths and formation of secondary pollen tubes. Both BET11 and BET12 are required for fertility and development of pollen tubes in Arabidopsis. More experiments are required to dissect the mechanisms involved. Academia Sinica (Taiwan) National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA), Taiwan National Science Council (NSF; 99-2321-B-001-036-MY3), Taiwan Universidad de Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM)

Deficiencies in boron, a microelement that is essential for the growth and development of higher plants, often cause problems in reproductive growth. Rhamnogalacturonan-II (RG-II) in cell wall pectin acts as the sole receptor for boron in plant cells, forming a borate cross-linked RG-II dimer (dRG-II-B), but the physiological functions of dRG-II-B remain unknown. We have previously shown that the pectin glucuronyltransferase 1 gene NpGUT1 , which is involved in the biosynthesis of RG-II sugar chains, is essential for the formation of the RG-II-B complex, resulting in tight intercellular attachment in meristematic tissues. Because NpGUT1 expression was found to be abundant in reproductive organs in addition to meristematic tissues, we analyzed the expression and functions of NpGUT1 in more detail in tobacco reproductive tissues. Specific NpGUT1 expression was detected in the tapetum of flower buds and in the pollen, pollen tube tips, and transmitting tissue of the pistils of flowers. Dexamethasone-induced expression of the NpGUT1 antisense gene in flower buds resulted in the formation of sterile flowers with aberrant development of pollen and transmitting tissue. Pollen tubes could not pass through pistils with aborted transmitting tissue, and expression of an NpGUT1 antisense gene in germinating pollen inhibited pollen tube elongation, accompanied by the absence of pectin RG-II and boron in the pollen tube tip. These results indicate that expression of NpGUT1 is required for the development and functions of male and female tissues.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein–tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.

The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.

With semi in vivo pollen tube growth assays, stigmas are pollinated in vivo and, after a fixed time interval, the styles are isolated from the ovary and placed on culture medium in vitro. Semi in vitro pollination includes isolation of the stigma and style complex, followed by pollination and placing the stylar end on nutrient medium. After semi in vivo pollination more and longer pollen tubes protruded from the cut end of the styles into medium, in comparison to semi in vitro pollination. Medium with 3 g l−1 agar was better than that with 6 g l−1 agar for pollen tube growth after the tubes emerged from the cut style. Semi in vitro pollination of the reversed style indicated that pollen tube growth was not influenced by the direction of the style. Fructose and glucose inhibited pollen tube growth compared to sucrose. Swollen tips characterized tube growth inhibition. After semi in vivo pollination all generative nuclei had divided to give two sperm nuclei. The average distance between the last sperm nucleus and the pollen tube tip as well as the distance between the two sperm nuclei diminished in growing pollen tubes between 24 and 48 h after pollination. The arrangements between the vegetative and the generative nuclei did not differ in semi in vivo and in vitro cultured pollen tubes of Aechmea fasciata. This information is important to explain why fertilization rate is low after placental pollination in comparison to placental grafted style pollination of Aechmea fasciata. The data may also contribute to the improvement of in vitro fertilization methods in Bromeliaceae and other higher plants.